Use of endoglin polypeptides for modifying the regulatory activity of TGF-β

ABSTRACT

This invention provides a novel purified TGF-β-binding glycoprotein, endoglin, an isolated nucleic acid molecule that encodes an amino acid sequence corresponding to the TGF-β-binding glycoprotein, soluble endoglin-derived polypeptide, and fragments thereof. A pharmaceutical composition which comprises the endoglin-derived polypeptide purified by applicants or produced by applicants&#39; recombinant methods and a pharmaceutically acceptable carrier is further provided as well as methods of treating patients which comprise administering to the patient the pharmaceutical composition of this invention.

This invention was made in part with Government support under Grant Nos.CA-08748 and CA-34610, from the National Institutes of Health. TheGovernment may have certain rights in this invention.

This application is a continuation of application Ser. No. 07/968,953,filed Oct. 30, 1992.

FIELD OF THE INVENTION

The present invention relates to cell biology and to methods ofmodifying the biological activity of cell regulatory factors. Morespecifically, the present invention relates to a novel TGF-β-bindingglycoprotein.

Throughout this application various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

BACKGROUND OF THE INVENTION

Glycoproteins, in which one or more carbohydrate units have beenattached covalently to the protein by posttranslational processing arewidely distributed. Several secretory proteins, including theimmunoglobulins, are glycoproteins, as are most components of plasmamembranes such as cell membrane receptors, where the carbohydrates canbe involved in cell-to-cell adhesion.

Transforming growth factor β (TGF-β) refers to a family ofmulti-functional cell regulatory factors produced in various forms bymany cell types (for review see Sporn et al,. J. Cell Biol., 105: 1039(1987)). Five distinct isoforms of TGF-β have been identified. TGF-β1and TGF-β2 have been characterized in detail. TGF-β is the subject ofU.S. Pat. Nos. 4,863,899; 4,816,561 and 4,742,003 which are incorporatedherein by reference. TGF-β binds to cell surface receptors present onvarious types of cells. TGF-β potentiates or inhibits the response ofmost cells to other growth factors, depending on the cell type. TGF-βalso regulates differentiation of some cell types, either promoting orinhibiting proliferation of the cell. A marked effect of TGF-β is thepromotion of cellular production of extracellular matrix proteins andtheir receptors (for a review see Keski-Oja et al., J. Cell Biochem.,33: 95 (1987); Massague, Cell 49: 437 (1987); Roberts and Sporn,"Peptides Growth Factors and Their Receptors", Springer-Verlag (1989)).

Notwithstanding the beneficial and essential cell regulatory functionsserved, TGF-β regulatory activity can prove detrimental to its hostorganism. For example, whereas growth and proliferation of mesenchymalcells is stimulated by TGF-β, some tumor cells may also be stimulated,using TGF-β as an autocrine growth factor. In other cases the inhibitionof cell proliferation by TGF-β similarly proves detrimental to its hostorganism. An example would be the prevention of new cell growth toassist in repair of tissue damage. The stimulation of extracellularmatrix production by TGF-β is essential for wound healing. However, insome cases, the TGF-β response is uncontrolled and an excessiveaccumulation of extracellular matrix results. An example of excessiveaccumulation of extracellular matrix is glomerulonephritis and scartissue formation.

The transforming growth factor-β receptor system in most mesenchymal andepithelial cells consists of several components (Massague, J. Ann. Rev.Cell Biol., 6: 597 (1990); Lin, H. Y. et al., Cell, 68: 775 (1992);Georgi, L. L. et al., Cell, 61: 635 (1990); Mathews, L. S. et al., Cell,65: 973 (1991); Attisano, L. et al., Cell. 68: 97 (1992); Lopez-Casillaset al., Cell, 67: 785 (1991) and Wang et al., Cell, 67: 796 (1991) allof which are incorporated herein by reference), one of which isbetaglycan, a membrane-anchored proteoglycan. In addition to betaglycan,the TGF-β receptor system in most mesenchymal and epithelial cellsconsists of the type I receptor, a 53-kDa glycoprotein whose structurehas not been determined yet, and the type II receptor, which belongs tothe protein serine/threonine kinase receptor family. Additional cellsurface TGF-β-binding proteins, some of which have a more restricteddistribution, have also been described.

Thus, a need exists to develop compounds that can modify the effects ofcell regulatory factors such as TGF-β. The present invention satisfiesthis need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a novel purified TGF-β-bindingglycoprotein. This novel human protein, endoglin, is expressed at highlevels on human vascular endothelial cells.

Further provided by the present invention are methods of treatingpathologic conditions mediated by TGF-β regulatory activity bycontacting the TGF-β with an effective amount of purifiedendoglin-derived polypeptide or any fragment thereof having the abilityto bind TGF-β. Thus, intact, native endoglin and soluble fragmentsthereof are useful in these methods. This invention provides a method ofpreparing and purifying soluble endoglin-derived polypeptide. Isolatednucleic acids encoding the novel TGF-β-binding glycoprotein and solubleendoglin-derived polypeptides are also provided, as well as vectorscontaining the nucleic acids and recombinant host cells transformed withsuch vectors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the domain structures of betaglycan and endoglin. Shown isa schematic representation highlighting regions of similarity betweenthe linear sequences of betaglycan, an 853-amino acid transmembraneproteoglycan, and endoglin, a disulfide-linked transmembrane proteincomposed of two identical subunits of 633 amino acids each. Thetransmembrane and short cytoplasmic regions (dark shaded box) ofendoglin have a high level of sequence similarity to the correspondingregions of betaglycan. Two regions of weaker similarity are detected inthe ectodomains of these proteins (light shaded boxes). Numbersrepresent the percent amino acid sequence similarity between theindicated domains of betaglycan and endoglin. Closed ovals representpositions of cysteine residues. Two putative sites for glycosaminoglycanchain attachment in betaglycan are indicated.

FIG. 2 shows cell surface TGF-β1-binding proteins expressed by HUVEC.Near confluent cultures of HUVECs were affinity-labeled by incubationwith 100 pM ¹²⁵ I-TGF-β1 followed by chemical cross-linking with 0.16 mMdisuccinimidyl suberate. A) Triton X-100 extracts of affinity-labeledHUVEC were resolved on SDS-PAGE gels under reducing (R) or nonreducing(NR) conditions. Lane C contains extract from cells affinity-labeled inthe presence of excess unlabeled TGF-β1. The migration position of TGF-βreceptors I (RI) and II (RII) are indicated. Arrow, the majoraffinity-labeled proteins of 180 kDa and higher molecular mass apparenton nonreducing gels. Arrowhead, the affinity-labeled proteins of 110-120kDa seen on reducing gels. B) detergent extracts of affinity-labeledHUVEC were resolved under nonreducing conditions on a first gel that wasthen resolved under reducing conditions in the second dimension aspreviously described. The 110-120-kDa labeled species migratingoff-the-diagonal are indicated (arrowheads).

FIG. 3 shows specific immunoprecipitation of TGF-β1-endoglin complexes.HUVECs were affinity-labeled with 100 pM ¹²⁵ I-TGF-β1 as described inFIG. 1. A) detergent extracts of affinity-labeled cells were incubatedwith mAb 44G4 and immune complexes were collected on proteinG-Sepharose. After washes, equal aliquots of the samples were analyzedunder reducing (R) or non-reducing (NR) conditions by SDS-PAGE (5-8%polyacrylamide gradient gels). B) affinity-labeled HUVEC lysates weremaximally depleted of endoglin by two successive 45 min incubations at4° C. with 100 μl of 44G4-IgG-Sepharose. S) supernatant after secondimmunoprecipitation. I) the first 44G4 immunoprecipitation whichcontained 83% of the endoglin. T) corresponding amount of total extractused for the depletion experiment. All samples were analyzed undernonreducing conditions on SDS-PAGE with the exception of I_(R), whichwas run under reducing conditions. The migration positions of TGF-βreceptor II (RII), and endoglin monomer, dimer, and oligomer areindicated.

FIG. 4 shows that endoglin transiently expressed in COS-M6 cells bindsTGF-β1. COS-M6 cells were transletted with a cDNA encoding full-lengthendoglin (Endoglin) or control vector (C). Cells were affinity-labeledwith 150 pM ¹²⁵ I-TGF-β1 and the detergent extracts incubated with mAb44G4 followed by protein G-Sepharose. Immunoprecipitated proteins wereanalyzed by SDS-PAGE under reducing (R) and nonreducing (NR) conditionsand visualized by autoradiography.

FIG. 5 shows the specificity of endoglin for TGF-β isoforms assessed inCOS cell transfectants and in HUVEC. A) COS-M6 cells transfected withendoglin vector were affinity-labeled with 150 pM ¹²⁵ I-TGF-β1 alone orin the presence of 1 or 10 nM unlabeled TGF-β1, -β2 or -β3. B) HUVECwere affinity-labeled with 100 pM ¹²⁵ I-TGF-β1 alone or in the presenceof 5 nM unlabeled TGF-β1 or TGF-β2. Lysates from these cells wereimmunoprecipitated with MAb 44G4. Immunoprecipitates were fractionatedunder reducing conditions on SDS-PAGE gels. The region of the gelscontaining monomeric endoglin is shown along with the migration positionof 100-kDa marker.

DETAILED DESCRIPTION OF THE INVENTION

Endoglin is a homodimeric membrane glycoprotein composed ofdisulfide-tinked subunits of 95 kDa. It is expressed in humanpre-erythroblasts, macrophages, leukemic cells of the lymphoid andmyeloid lineages, and at higher levels in syncytiotrophoblast oftermplacenta and vascular endothelial cells. A relationship betweenhuman endoglin and the TGF-β receptor system was discovered with themolecular cloning of the rat TGF-β-binding proteoglycan, betaglycan(also known as the type III TGF-β receptor), which revealed that thetransmembrane domain and the relatively short (43 amino acid)cytoplasmic tail of this protein were remarkably similar (71% amino acidsequence similarity and 63% amino acid identity) to the correspondingregions in endoglin (see FIG. 1). The extracellular domains of these twoproteins show limited homology in primary structure, and while endoglinis not a proteoglycan, it does contain N- and O-linked oligosaccharides.

The sequence of endoglin revealed a Type I integral membrane protein of68,051 Daltons. The extracellular region of 561 amino acids contains 4potential N-linked glycosylation sites and an O-glycan domain rich inserine and threonine residues proximal to the plasma membrane: a singlehydrophobic transmembrane region is followed by a 47-amino acidcytoplasmic tail. The presence of an Arg-Gly-Asp (RGD) motif in anaccessible region of the polypeptide led to the suggestion that thisintegral membrane protein may play a role in RGD-mediated cellularadhesion events. Endogtin contains an RGD sequence and so is potentiallyinvolved in RGD-mediated cellular adhesion, whereas betaglycan does notcontain this sequence.

Accordingly, the present invention provides a soluble endoglin-derivedpolypeptide that binds TGF-β. The full-length soluble endoglin-derivedpolypeptide comprises the 561 amino acids of the extracellular domain ofthe mature endoglin polypeptide, an integral membrane protein, whichconsists of 633 amino acids in total. The nucleic acid sequence encodingthe 633 amino acid mature endoglin polypeptide is identified in SEQ IDNO: 1-2. The nucleic acid sequence encoding the soluble endoglin-derivedpolypeptide is included within the sequence set forth in SEQ ID NO: 1(from about amino acid number 1 to about amino acid number 561).

As used herein, the term "purified" means that the molecule or compoundis substantially free of contaminants normally associated with a nativeor natural environment. For example, the mature 633 amino acid proteincan be obtained from a number of methods. The methods available for thepurification of membrane proteins include precipitation, gel filtration,ion-exchange, reversed-phase, and affinity chromatography. Otherwell-known methods are described in Deutscher et al., Guide to ProteinPurification: Methods in Enzymology Vol. 182, (Academic Press 1990),which is incorporated herein by reference. Alternatively, a purifiedpolypeptide of the present invention can also be obtained by well-knownrecombinant methods as described, for example, in Maniatis et al.,Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring HarborLaboratory 1989), also incorporated herein by reference. An example ofthis means for preparing soluble endoglin-derived polypeptide is toexpress nucleic acid encoding the soluble endoglin in a suitable hostcell, such as a bacterial, yeast or mammalian cell, using methods wellknown in the art, and recovering the expressed soluble protein, againusing methods well known in the art. The soluble polypeptide andbiologically active fragments thereof can also be produced by chemicalsynthesis. Synthetic polypeptides can be produced using AppliedBiosystems, Inc. Model 430A or 431A automatic polypeptide synthesizerand chemistry provided by the manufacturer. The soluble polypeptide canalso be isolated directly from cells which have been transformed withthe expression vectors described below in more detail.

As used herein, endoglin-derived polypeptide means a polypeptide havingthe amino acid sequence substantially the same as the 633 amino acidsequence shown in SEQ ID NO: 2, or an active fragment thereof. As usedherein the term "soluble endoglin-derived polypeptide" refers to asoluble, biologically active fragment of the human endoglin polypeptideexpressed by the extracellular domain of the nucleic acid. As usedherein, an "active fragment" or "biologically-active fragment" refers toany portion of the endoglin polypeptide shown in SEQ ID NO: 2 that bindto TGF-β. Methods of determining whether a polypeptide can bind TGF-βare well known to those of skill in the art, for example, as set forthherein.

The invention also encompasses nucleic acid molecules which differ fromthat of the nucleic acid molecule shown in SEQ ID NO: 1, but whichproduce the same phenotypic effect. These altered, but phenotypicallyequivalent nucleic acid molecules are referred to as "equivalent nucleicacids". This invention also encompasses nucleic acid moleculescharacterized by changes in non-coding regions that do not alter thephenotype of the polypeptide produced therefrom when compared to thenucleic acid molecule described hereinabove. This invention furtherencompasses nucleic acid molecules which hybridize to the nucleic acidmolecule of the subject invention. As used herein, the term "nucleicacid" encompasses RNA as well as single and double-stranded DNA andcDNA. In addition, as used herein, the term "polypeptide" encompassesany naturally occurring allelic variant thereof as well as man-maderecombinant forms.

This invention provides an isolated nucleic acid molecule encoding ahuman soluble endoglin-derived polypeptide. As used herein, the term"isolated nucleic acid molecule" means a nucleic acid molecule that isin a form that does not occur in nature. One means of isolating a humanendoglin nucleic acid is to probe a human cDNA expression library with anatural or artificially designed antibody to endoglin, using methodswell known in the art (see Gougos, A. et al., J. Biol Chem., 265: 8361(1990) which is incorporated herein by reference). DNA and cDNAmolecules which encode human endoglin polypeptides can be used to obtaincomplementary genomic DNA, cDNA or RNA from human, mammalian or otheranimal sources.

The invention further provides the isolated nucleic acid moleculeoperatively linked to a promoter of RNA transcription, as well as otherregulatory sequences. As used herein, the term "operatively linked"means positioned in such a manner that the promoter will direct thetranscription of RNA off of the nucleic acid molecule. Examples of suchpromoters are SP6, T4 and T7. Vectors which contain both a promoter anda cloning site into which an inserted piece of DNA is operatively linkedto that promoter are well known in the art. Preferably, these vectorsare capable of transcribing RNA in vitro or in vivo. Examples of suchvectors are the pGEM series (Promega Biotec, Madison, Wis.).

This invention provides a vector comprising this isolated nucleic acidmolecule such as DNA, cDNA or RNA encoding a soluble endoglin-derivedpolypeptide. Examples of vectors are viruses, such as bacteriophages,baculoviruses and retroviruses, cosmids, plasmids (such as pcEXV-2) andother recombination vectors. Nucleic acid molecules are inserted intovector genomes by methods well known in the art. For example, insert andvector DNA can both be exposed to a restriction enzyme to createcomplementary ends on both molecules that base pair with each other andwhich are then joined together with a ligase. Alternatively, syntheticnucleic acid linkers can be ligated to the insert DNA that correspond toa restriction site in the vector DNA, which is then digested with arestriction enzyme that recognizes a particular nucleotide sequence.Additionally, an oligonucleotide containing a termination codon and anappropriate restriction site can be ligated for insertion into a vectorcontaining, for example, some or all of the following: a selectablemarker gene, such as neomycin gene for selection of stable or transienttransfectants in mammalian cells; enhancer/promoter sequences from theimmediate early gene of human CMV for high levels of transcription;transcription termination and RNA processing signals from SV40 for mRNAstability; SV40 polyoma origins of replication and ColE1 for properepisomal replication; versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and anti-sense RNA.Other means are available.

Also provided are vectors comprising a DNA molecule encoding a humansoluble endoglin-derived polypeptide, adapted for expression in abacterial cell, a yeast cell, a mammalian cell and other animal cells.The vectors additionally comprise the regulatory elements necessary forexpression of the DNA in the bacterial, yeast, mammalian or animal cellsso located relative to the DNA encoding soluble endoglin polypeptide asto permit expression thereof. Regulatory elements required forexpression include promoter sequences to bind RNA polymerase andtranscription initiation sequences for ribosome binding. For example, abacterial expression vector includes a promoter such as the lac promoterand for transcription initiation the Shine-Dalgarno sequence and thestart codon AUG (Maniatis et al. supra. 1989). Similarly, a eucaryoticexpression vector includes a heterologous or homologous promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon AUG,and a termination codon for detachment of the ribosome. Such vectors canbe obtained commercially or assembled by the sequences described inmethods well known in the art, for example the methods described abovefor constructing vectors in general. Expression vectors are usefulproduce cells that express the polypeptide.

This invention provides a mammalian cell containing a cDNA moleculeencoding a human soluble endoglin-derived polypeptide. An example is amammalian cell comprising a plasmid adapted for expression in amammalian cell. The plasmid has a cDNA molecule encoding a solubleendoglin-derived polypeptide and the regulatory elements necessary forexpression of the polypeptide. Various mammalian cells may be utilizedas hosts, including, for example, mouse fibroblast cell NIH3T3, CHOcells, HeLa cells, Ltk- cells, etc. Expression plasmids such as thosedescribed supra can be used to transfect mammalian cells by methods wellknown in the art such as calcium phosphate precipitation, DEAE-dextran,electropotation or microinjection.

This invention provides a pharmaceutical composition containing apharmaceutical carrier and any of a purified, soluble polypeptide, anactive fragment thereof, or a purified, mature protein and activefragments thereof, alone or in combination with each other. Thesepolypeptides or proteins can be recombinantly derived, chemicallysynthesized or purified from native sources. As used herein, the term"pharmaceutically acceptable carrier" encompasses any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water and emulsions such as an oil/water or water/oil emulsion, andvarious types of wetting agents.

Also provided are antibodies having specific reactivity with theendoglin-derived TGF-β-binding polypeptides of the subject invention,such as anti-endoglin antibody 44G4, or any antibody having specificreactivity to a TGF-β-binding endoglin polypeptide. Active fragments ofantibodies are encompassed within the definition of "antibody." Theantibodies of the invention can be produced by any method known in theart. For example, polyclonal and monoclonal antibodies can be producedby methods well known in the art, as described, for example, in Harlowand Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory1988), which is incorporated herein by reference. The polypeptide,particularly soluble endoglin-derived polypeptide of the presentinvention, can be used as the immunogen in generating such antibodies.Altered antibodies, such as chimetic, humanized, CDR-grafted orbifunctional antibodies can also be produced by methods well known tothose skilled in the art. Such antibodies can also be produced byhybridoma, chemical synthesis or recombinant methods described, forexample, in Maniatis et al., supra, incorporated herein by reference.The antibodies can be used for determining the presence or purificationof the soluble endoglin-derived polypeptide of the present invention.With respect to the detecting of such polypeptides, the antibodies canbe used for in vitro diagnostic or in vivo imaging methods.

Immunological procedures useful for in vitro detection of the targetsoluble endoglin-derived polypeptide in a sample include immunoassaysthat employ a detectable antibody. Such immunoassays include, forexample, ELISA, Pandex microfluorimetric assay, agglutination assays,flow cytometry, serum diagnostic assays and immunohistochemical stainingprocedures which are well known in the art. An antibody can be madedetectable by various means well known in the art. For example, adetectable marker can be directly or indirectly attached to theantibody. Useful markers include, for example, radionuclides, enzymes,fluorogens, chromogens and chemiluminescent labels.

This invention provides a method of modifying a biological functionmediated by the regulatory activity of TGF-β which comprises contactinga suitable sample containing TGF-β with an effective amount of abiologically active endoglin-derived polypeptide or a pharmaceuticalcomposition described above. As used herein, "an effective amount"refers to an amount of the polypeptide sufficient to bind to TGF-β andthereby prevent or inhibit its regulatory activity. This method isespecially useful for modifying the regulatory activity of TGF-β1 orTGF-β3. Examples of regulatory activities include, but are not limitedto stimulation of cell proliferation, cell growth inhibition, orpromotion of extracellular matrix proteins.

An effective amount is any amount that is effective to modify thebiological function mediated by the regulatory activity of TGF-β. Themethod can be practiced in vitro or in vivo. If the method is practicedin vitro, contacting is effected by incubating the sample with apolypeptide, a protein or a pharmaceutical composition as describedabove.

However, in a preferred embodiment the contacting is effected in vivo byadministering a polypeptide, a protein or a pharmaceutical composition,as described above, to a subject, e.g., a human patient.

Methods of administration are well known to those of skill in the artand include, but are not limited to administration orally, intravenouslyor parenterally. Administration will be in such a dosage such that theregulatory activity is effectively modified. Administration can beeffected continuously or intermittently such that this amount iseffective for its intended purpose.

This invention also provides a method of treating a pathologic conditioncaused by a TGF-β-regulated activity comprising contacting the TGF-βwith any of a purified soluble endoglin-derived polypeptide, an activefragment thereof, an endoglin-derived polypeptide or an active fragmentthereof. The TGF-β is bound with said polypeptide to thereby treat thepathologic condition mediated by TGF-β regulatory activity. As usedherein, "pathologic conditions" refers to any pathology arising fromTGF-β-induced regulatory activity. For example, growth and proliferationof mesenchymal cells is stimulated by TGF-β, however some tumor cellsmay also be stimulated thus using TGF-β as an autocrine growth factor.An example of inhibitory conditions are the prevention of new cellgrowth to assist in repair of tissue damage. The stimulation ofextracellular matrix production by TGF-β is essential for wound healing.However, in some cases, the TGF-β response is uncontrolled and anexcessive accumulation of extracellular matrix results. An example ofexcessive accumulation of extracellular matrix is glomerulonephritis.Additional examples of pathologies include cancer, rheumatoid arthritisand atherosclerosis.

In a preferred embodiment, the method is practiced by administering to asubject, e.g., a human patient, an effective amount of a purifiedendoglin protein or an endogtin-derived soluble polypeptide or abiologically active fragment thereof, or the pharmaceutical compositiondescribed above. Methods of administration are outlined supra.

It is understood that modifications which do not substantially affectthe activity of the various molecules of this invention are alsoincluded within the definition of said molecules.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Cell Culture and Transfections

Human umbilical vein endothelial cells (HUVEC, CRL 1730, ATCC) weremaintained in α-minimal essential media supplemented according tosupplier's instructions or prepared from umbilical veins as previouslydescribed (Gougos, A. et al., J.Immunol., 141: 1925 (1988)). Similarresults were obtained using cells from either source. COS-M6 cells,maintained in Dulbecco's modified Eagle's medium supplemented with 10%bovine serum, were transfected with a cDNA encoding full-length endoglinligated into the EcoRI site of the mammalian expression vector pcEXV(Miller, J. et al., J.Exp.Med., 164: 1478 (1986)) or with a controlvector without cDNA insert (pcMV5; Lopez-Casillas, F. et al., Cell, 67:785 (1991)) by the DEAE-dextran-chloroquine procedure (Seed, B., et al.,Proc.Natl.Acad.Sci. USA, 84: 3365 (1987)). 24 hours post-transfection,cells were trypsinized and reseeded into multicluster dishes and allowedto grow an additional 48 hours before being affinity-labeled with ¹²⁵I-TGF-β1 as described below.

EXAMPLE II Receptor Affinity Labeling and Immunoprecipitation

TGF-β1 and TGF-β2 were purchased from R & D Systems (Minneapolis, Minn.)and TGF-β3 was obtained from Oncogene Science (Manhassett, N.Y.). ¹²⁵I-TGF-β1 used in these studies was prepared by the chloramine-T methodas previously described (Cheifetz, S. et al., J.Biol.Chem., 265: 20533(1990)) or purchased from Amersham Corp.; both preparations gaveidentical results. The conditions for affinity labeling cell monolayerswith ¹²⁵ I-TGF-β1 and disuccinimidyl suberate (Pierce Chemical Co.) havebeen described previously (Massague, J., Methods Enzymol., 146: 174(1987)). The concentrations of ¹²⁵ I-TGF-β1 and competing unlabeledligands used for each experiment are indicted in the figure legends.Triton X-100 extracts of the affinity-labeled cells were either analyzeddirectly on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) orfirst incubated with monoclonal antibody (mAb) 44G4 directed againsthuman endoglin (Quackenbush, E. J. et al., J.Immunol., 134: 1276 (1985))or with control antibody (see below). For immunoprecipitations,detergent extracts were diluted with an equal volume ofphosphate-buffered saline containing 1% Triton X-100 and precleared byincubation for 20 min at 4° C. with protein G-Sepharose (Pharmacia LKBBiotechnology Inc.) prior to overnight incubation at 4° C. with mAb44G4. Immune complexes were collected by incubation with proteinG-Sepharose for 1 hour at 4° C. For some experiments, mAb 44G4 was usedcoupled to Sepharose. The immunoprecipitates were washed three times(saline with 1% Triton X-100) and then resolved by SDS-PAGE in thepresence or absence of dithiothreitol (DTT) and visualized byautoradiography. Irrelevant mAb (44D7) used in control experiments tomonitor specificity of the immunoprecipitations did notimmunoprecipitate any affinity-labeled bands.

EXAMPLE III SDS-PAGE and 2D-Gel Analysis

Analysis of the affinity-labeled profile of HUVEC revealed that, likevascular endothelial cells from other sources, these cells have littleor no betaglycan, which characteristically migrates as a diffuse bandbetween 200 and 400 KDa on reducing SDS-PAGE (FIG. 2A). Instead, HUVECexpressed a disulfide-linked cell surface protein that, together withTGF-β receptors I and II, was affinity-labeled by crosslinking with ¹²⁵I-TGF-β1. Receptors I and II were detected in HUVEC as labeled complexesof approximately 65 KDa and 100 KDa, which is similar to the size ofthese labeled receptors reported for other human cell lines. Comparisonof the relative migration of the affinity-labeled proteins fractioned onSDS-PAGE revealed that the major affinity-labeled proteins of HUVECmigrated between 95-120 KDa on reducing gels whereas on non-reducinggels the major affinity-labeled proteins migrated between 100-110 KDa(presumed to be receptor II) and at 180 KDa and above (endoglin) (FIG.2). This pattern indicated the presence of disulfide-tinkedTGF-β-binding proteins.

Resolution of these disulfide-linked TGF-β1 binding proteins ontwo-dimensional gels (FIG. 2B) confirmed that the disulfide-tinkedcomplexes (probably dimers and higher order oligomers) containedsubunits of approximately 95 KDa (value estimated by subtracting thecross-linked TGF-β1 monomer mass 12.5 KDa from the reduced 110 KDaaffinity-labeled complex). Together with the type II receptor, thedisulfide-linked TGF-β1-binding proteins are the major affinity-labeledspecies expressed by HUVEC.

EXAMPLE IV Immunoprecipitation with anti-endoglin mAb

To determine whether the disulfide-linked TGF-β-binding protein onendothelial cells was endoglin, affinity-labeled HUVEC extracts wereimmunoprecipitated with monoclonal antibody (mAb) 44G4 which is specificfor human endoglin (Georgi, L. L. et al., Cell, 61: 635 (1990); MacKay,K. et al., J.Biol.Chem., 266: 9907 (1992); Merwin, J. R. et al.,Am.J.Pathol., 138: 37 (1991)). Electrophoretic analysis of theseimmunoprecipitates revealed a labeled protein complex whose subunitstructure was similar to that of endoglin (FIG. 3A). Thus, underreducing conditions, a major affinity-labeled band of approximately 110KDa was seen which migrated as complexes of 180 KDa and greater than 200KDa when analyzed under non-reducing conditions. The higher orderoligomers might contain multiple endoglin molecules crosslinked byTGF-β1, itself a disulfide-linked dimer. Repeated immunoprecipitationwith 44G4-IgG-Sepharose completely depleted these labeled species fromcell extracts (FIG. 3B). No affinity-labeled bands wereimmunoprecipitated from three other human cell lines (A549, Hep G2,MCF-7), which lack endoglin and were used as negative-controls for theseexperiments. Monoclonal antibodies specific to human endoglin and anendoglin expression vector thus demonstrate that endoglin is a majorTGF-β-binding protein in human vascular endothelial cells.

EXAMPLE V Ectopic Expression of Endoglin in Cells

The identity of this dimeric TGF-β-binding protein of HUVEC withendoglin was confirmed by ectopically expressing the full-lengthendoglin cDNA in COS monkey kidney cells. After affinity-labeling with¹²⁵ I-TGF-β1, a labeled species with the characteristics of endoglincould be specifically precipitated by mAb 44G4 only from the detergentextracts of endoglin transfectants (FIG. 4). Differences inglycosylation likely account for the smaller size of endoglin expressedin COS cells relative to endogenous endoglin of HUVEC.

Although the invention has been described with reference to thedisclosed embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2620 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1935                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGGGCCAGCTGCAGCCTCAGCCCCACAAGTCTTGCAGAAACAGTCCAT48                            GlyAlaSerCysSerLeuSerProThrSerLeuAlaGluThrValHis                              151015                                                                        TGTGACCTTCAGCCTGTGGGCCCCGAGAGGGGCGAGGTGACATATACC96                            CysAspLeuGlnProValGlyProGluArgGlyGluValThrTyrThr                              202530                                                                        ACTAGCCAGGTCTCGAAGGGCTGCGTGGCTCAGGCCCCCAATGCCATC144                           ThrSerGlnValSerLysGlyCysValAlaGlnAlaProAsnAlaIle                              354045                                                                        CTTGAAGTCCATGTCCTCTTCCTGGAGTTCCCAACGGGCCCGTCACAG192                           LeuGluValHisValLeuPheLeuGluPheProThrGlyProSerGln                              505560                                                                        CTGGAGCTGACTCTCCAGGCATCCAAGCAAAATGGCACCTGGCCCCGA240                           LeuGluLeuThrLeuGlnAlaSerLysGlnAsnGlyThrTrpProArg                              65707580                                                                      GAGGTGCTTCTGGTCCTCAGTGTAAACAGCAGTGTCTTCCTGCATCTC288                           GluValLeuLeuValLeuSerValAsnSerSerValPheLeuHisLeu                              859095                                                                        CAGGCCCTGGGAATCCCACTGCACTTGGCCTACAATTCCAGCCTGGTC336                           GlnAlaLeuGlyIleProLeuHisLeuAlaTyrAsnSerSerLeuVal                              100105110                                                                     ACCTTCCAAGAGCCCCCGGGGGTCAACACCACAGAGCTGCCATCCTTC384                           ThrPheGlnGluProProGlyValAsnThrThrGluLeuProSerPhe                              115120125                                                                     CCCAAGACCCAGATCCTTGAGTGGGCAGCTGAGAGGGGCCCCATCACC432                           ProLysThrGlnIleLeuGluTrpAlaAlaGluArgGlyProIleThr                              130135140                                                                     TCTGCTGCTGAGCTGAATGACCCCCAGAGCATCCTCCTCCGACTGGGC480                           SerAlaAlaGluLeuAsnAspProGlnSerIleLeuLeuArgLeuGly                              145150155160                                                                  CAAGCCCAGGGGTCACTGTCCTTCTGCATGCTGGAAGCCAGCCAGGAC528                           GlnAlaGlnGlySerLeuSerPheCysMetLeuGluAlaSerGlnAsp                              165170175                                                                     ATGGGCCGCACGCTCGAGTGGCGGCCGCGTACTCCAGCCTTGGTCCGG576                           MetGlyArgThrLeuGluTrpArgProArgThrProAlaLeuValArg                              180185190                                                                     GGCTGCCACTTGGAAGGCGTGGCCGGCCACAAGGAGGCGCACATCCTG624                           GlyCysHisLeuGluGlyValAlaGlyHisLysGluAlaHisIleLeu                              195200205                                                                     AGGGTCCTGCCGGGCCACTCGGCCGGGCCCCGGACGGTGACGGTGAAG672                           ArgValLeuProGlyHisSerAlaGlyProArgThrValThrValLys                              210215220                                                                     GTGGAACTGAGCTGCGCACCCGGGGATCTCGATGCCGTCCTCATCCTG720                           ValGluLeuSerCysAlaProGlyAspLeuAspAlaValLeuIleLeu                              225230235240                                                                  CAGGGTCCCCCCTACGTGTCCTGGCTCATCGACGCCAACCACAACATG768                           GlnGlyProProTyrValSerTrpLeuIleAspAlaAsnHisAsnMet                              245250255                                                                     CAGATCTGGACCACTGGAGAATACTCCTTCAAGATCTTTCCAGAGAAA816                           GlnIleTrpThrThrGlyGluTyrSerPheLysIlePheProGluLys                              260265270                                                                     AACATTCGTGGCTTCAAGCTCCCAGACACACCTCAAGGCCTCCTGGGG864                           AsnIleArgGlyPheLysLeuProAspThrProGlnGlyLeuLeuGly                              275280285                                                                     GAGGCCCGGATGCTCAATGCCAGCATTGTGGCATCCTTCGTGGAGCTA912                           GluAlaArgMetLeuAsnAlaSerIleValAlaSerPheValGluLeu                              290295300                                                                     CCGCTGGCCAGCATTGTCTCACTTCATGCCTCCAGCTGCGGTGGTAGG960                           ProLeuAlaSerIleValSerLeuHisAlaSerSerCysGlyGlyArg                              305310315320                                                                  CTGCAGACCTCACCCGCACCGATCCAGACCACTCCTCCCAAGGACACT1008                          LeuGlnThrSerProAlaProIleGlnThrThrProProLysAspThr                              325330335                                                                     TGTAGCCCGGAGCTGCTCATGTCCTTGATCCAGACAAAGTGTGCCGAC1056                          CysSerProGluLeuLeuMetSerLeuIleGlnThrLysCysAlaAsp                              340345350                                                                     GACGCCATGACCCTGGTACTAAAGAAAGAGCTTGTTGCGCATTTGAAG1104                          AspAlaMetThrLeuValLeuLysLysGluLeuValAlaHisLeuLys                              355360365                                                                     TGCACCATCACGGGCCTGACCTTCTGGGACCCCAGCTGTGAGGCAGAG1152                          CysThrIleThrGlyLeuThrPheTrpAspProSerCysGluAlaGlu                              370375380                                                                     GACAGGGGTGACAAGTTTGTCTTGCGCAGTGCTTACTCCAGCTGTGGC1200                          AspArgGlyAspLysPheValLeuArgSerAlaTyrSerSerCysGly                              385390395400                                                                  ATGCAGGTGTCAGCAAGTATGATCAGCAATGAGGCGGTGGTCAATATC1248                          MetGlnValSerAlaSerMetIleSerAsnGluAlaValValAsnIle                              405410415                                                                     CTGTCGAGCTCATCACCACAGCGGAAAAAGGTGCACTGCCTCAACATG1296                          LeuSerSerSerSerProGlnArgLysLysValHisCysLeuAsnMet                              420425430                                                                     GACAGCCTCTCTTTCCAGCTGGGCCTCTACCTCAGCCCACACTTCCTC1344                          AspSerLeuSerPheGlnLeuGlyLeuTyrLeuSerProHisPheLeu                              435440445                                                                     CAGGCCTCCAACACCATCGAGCCGGGGCAGCAGAGCTTTGTGCAGGTC1392                          GlnAlaSerAsnThrIleGluProGlyGlnGlnSerPheValGlnVal                              450455460                                                                     AGAGTGTCCCCATCCGTCTCCGAGTTCCTGCTCCAGTTAGACAGCTGC1440                          ArgValSerProSerValSerGluPheLeuLeuGlnLeuAspSerCys                              465470475480                                                                  CACCTGGACTTGGGGCCTGAGGGAGGCACCGTGGAACTCATCCAGGGC1488                          HisLeuAspLeuGlyProGluGlyGlyThrValGluLeuIleGlnGly                              485490495                                                                     CGGGCGGCCAAGGGCAACTGTGTGAGCCTGCTGTCCCCAAGCCCCGAG1536                          ArgAlaAlaLysGlyAsnCysValSerLeuLeuSerProSerProGlu                              500505510                                                                     GGTGACCCGCGCTTCAGCTTCCTCCTCCACTTCTACACAGTACCCATA1584                          GlyAspProArgPheSerPheLeuLeuHisPheTyrThrValProIle                              515520525                                                                     CCCAAAACCGGCACCCTCAGCTGCACGGTAGCCCTGCGTCCCAAGACC1632                          ProLysThrGlyThrLeuSerCysThrValAlaLeuArgProLysThr                              530535540                                                                     GGGTCTCAAGACCAGGAAGTCCATAGGACTGTCTTCATGCGCTTGAAC1680                          GlySerGlnAspGlnGluValHisArgThrValPheMetArgLeuAsn                              545550555560                                                                  ATCATCAGCCCTGACCTGTCTGGTTGCACAAGCAAAGGCCTCGTCCTG1728                          IleIleSerProAspLeuSerGlyCysThrSerLysGlyLeuValLeu                              565570575                                                                     CCCGCCGTGCTGGGCATCACCTTTGGTGCCTTCCTCATCGGGGCCCTG1776                          ProAlaValLeuGlyIleThrPheGlyAlaPheLeuIleGlyAlaLeu                              580585590                                                                     CTCACTGCTGCACTCTGGTACATCTACTCGCACACGCGTTCCCCCAGC1824                          LeuThrAlaAlaLeuTrpTyrIleTyrSerHisThrArgSerProSer                              595600605                                                                     AAGCGGGAGCCCGTGGTGGCGGTGGCTGCCCCGGCCTCCTCGGAGAGC1872                          LysArgGluProValValAlaValAlaAlaProAlaSerSerGluSer                              610615620                                                                     AGCAGCACCAACCACAGCATCGGGAGCACCCAGAGCACCCCCTGCTCC1920                          SerSerThrAsnHisSerIleGlySerThrGlnSerThrProCysSer                              625630635640                                                                  ACCAGCAGCATGGCATAGCCCCGGCCCCCCGCGCTCGCCCAGCAGGAGAGACTGA1975                   ThrSerSerMetAla                                                               645                                                                           GCAGCCGCCAGCTGGGAGCACTGGTGTGAACTCACCCTGGGAGCCAGTCCTCCACTCGAC2035              CCAGAATGGAGCCTGCTCTCCGCGCCTACCCTTCCCGCCTCCCTCTCAGAGGCCTGCTGC2095              CAGTGCAGCCACTGGCTTGGAACACCTTGGGGTCCCTCCACCCCACAGAACCTTCAACCC2155              AGTGGGTCTGGGATATGGCTGCCCAGGAGACAGACCACTTGCCACGCTGTTGTAAAAACC2215              CAAGTCCCTGTCATTTGAACCTGGATCCAGCACTGGTGAACTGAGCTGGGCAGGAAGGGA2275              GAACTTGAAACAGATTCAGGCCAGCCCAGCCAGGCCAACAGCACCTCCCCGCTGGGAAGA2335              GAAGAGGGCCCAGCCCAGAGCCACCTGGATCTATCCCTGCGGCCTCCACACCTGAACTTG2395              CCTAACTAACTGGCAGGGGAGACAGGAGCCTAGCGGAGCCCAGCCTGGGAGCCCAGAGGG2455              TGGCAAGAACAGTGGGCGTTGGGAGCCTAGCTCCTGCCACATGGAGCCCCCTCTGCCGGT2515              CGGGCAGCCAGCAGAGGGGGAGTAGCCAAGCTGCTTGTCCTGGGCCTGCCCCTGTGTATT2575              CACCACCAATAAATCAGACCATGAAACCAGTGAAAAAAAAAAAAA2620                             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 645 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyAlaSerCysSerLeuSerProThrSerLeuAlaGluThrValHis                              151015                                                                        CysAspLeuGlnProValGlyProGluArgGlyGluValThrTyrThr                              202530                                                                        ThrSerGlnValSerLysGlyCysValAlaGlnAlaProAsnAlaIle                              354045                                                                        LeuGluValHisValLeuPheLeuGluPheProThrGlyProSerGln                              505560                                                                        LeuGluLeuThrLeuGlnAlaSerLysGlnAsnGlyThrTrpProArg                              65707580                                                                      GluValLeuLeuValLeuSerValAsnSerSerValPheLeuHisLeu                              859095                                                                        GlnAlaLeuGlyIleProLeuHisLeuAlaTyrAsnSerSerLeuVal                              100105110                                                                     ThrPheGlnGluProProGlyValAsnThrThrGluLeuProSerPhe                              115120125                                                                     ProLysThrGlnIleLeuGluTrpAlaAlaGluArgGlyProIleThr                              130135140                                                                     SerAlaAlaGluLeuAsnAspProGlnSerIleLeuLeuArgLeuGly                              145150155160                                                                  GlnAlaGlnGlySerLeuSerPheCysMetLeuGluAlaSerGlnAsp                              165170175                                                                     MetGlyArgThrLeuGluTrpArgProArgThrProAlaLeuValArg                              180185190                                                                     GlyCysHisLeuGluGlyValAlaGlyHisLysGluAlaHisIleLeu                              195200205                                                                     ArgValLeuProGlyHisSerAlaGlyProArgThrValThrValLys                              210215220                                                                     ValGluLeuSerCysAlaProGlyAspLeuAspAlaValLeuIleLeu                              225230235240                                                                  GlnGlyProProTyrValSerTrpLeuIleAspAlaAsnHisAsnMet                              245250255                                                                     GlnIleTrpThrThrGlyGluTyrSerPheLysIlePheProGluLys                              260265270                                                                     AsnIleArgGlyPheLysLeuProAspThrProGlnGlyLeuLeuGly                              275280285                                                                     GluAlaArgMetLeuAsnAlaSerIleValAlaSerPheValGluLeu                              290295300                                                                     ProLeuAlaSerIleValSerLeuHisAlaSerSerCysGlyGlyArg                              305310315320                                                                  LeuGlnThrSerProAlaProIleGlnThrThrProProLysAspThr                              325330335                                                                     CysSerProGluLeuLeuMetSerLeuIleGlnThrLysCysAlaAsp                              340345350                                                                     AspAlaMetThrLeuValLeuLysLysGluLeuValAlaHisLeuLys                              355360365                                                                     CysThrIleThrGlyLeuThrPheTrpAspProSerCysGluAlaGlu                              370375380                                                                     AspArgGlyAspLysPheValLeuArgSerAlaTyrSerSerCysGly                              385390395400                                                                  MetGlnValSerAlaSerMetIleSerAsnGluAlaValValAsnIle                              405410415                                                                     LeuSerSerSerSerProGlnArgLysLysValHisCysLeuAsnMet                              420425430                                                                     AspSerLeuSerPheGlnLeuGlyLeuTyrLeuSerProHisPheLeu                              435440445                                                                     GlnAlaSerAsnThrIleGluProGlyGlnGlnSerPheValGlnVal                              450455460                                                                     ArgValSerProSerValSerGluPheLeuLeuGlnLeuAspSerCys                              465470475480                                                                  HisLeuAspLeuGlyProGluGlyGlyThrValGluLeuIleGlnGly                              485490495                                                                     ArgAlaAlaLysGlyAsnCysValSerLeuLeuSerProSerProGlu                              500505510                                                                     GlyAspProArgPheSerPheLeuLeuHisPheTyrThrValProIle                              515520525                                                                     ProLysThrGlyThrLeuSerCysThrValAlaLeuArgProLysThr                              530535540                                                                     GlySerGlnAspGlnGluValHisArgThrValPheMetArgLeuAsn                              545550555560                                                                  IleIleSerProAspLeuSerGlyCysThrSerLysGlyLeuValLeu                              565570575                                                                     ProAlaValLeuGlyIleThrPheGlyAlaPheLeuIleGlyAlaLeu                              580585590                                                                     LeuThrAlaAlaLeuTrpTyrIleTyrSerHisThrArgSerProSer                              595600605                                                                     LysArgGluProValValAlaValAlaAlaProAlaSerSerGluSer                              610615620                                                                     SerSerThrAsnHisSerIleGlySerThrGlnSerThrProCysSer                              625630635640                                                                  ThrSerSerMetAla                                                               645                                                                           __________________________________________________________________________

We claim:
 1. A method of modulating a biological activity mediated byTGF-β, which method comprises contacting TGF-β which is present in saidsystem with an amount of a polypeptide comprising the extracellulardomain of endoglin, said amount effective to modulate said biologicalfunction.
 2. The method of claim 1, wherein TGF-β is TGF-β1.
 3. Themethod of claim 1, wherein TGF-β is TGF-β3.
 4. The method of claim 1,wherein the contacting is effected in vitro.
 5. The method of claim 1,wherein the contacting is effected in vivo.
 6. The method of claim 1,wherein the biological activity is stimulation of cell proliferation orcell growth inhibition.
 7. The method of claim 1, wherein the biologicalactivity is extracellular matrix production.
 8. The method of claim 1,wherein the polypeptide is a purified polypeptide comprising adisulfide-linked homodimer of 95 kDa subunits.
 9. The method of claim 1,wherein the polypeptide is a purified polypeptide having a molecularmass of 95 kDa.
 10. The method of claim 8 or 9, wherein the purifiedpolypeptide is a purified and isolated human protein substantially freeof other human proteins.
 11. The method of claim 1, wherein thepolypeptide is a soluble polypeptide.
 12. A method of treating apathologic condition caused by TGF-β regulated cell growth stimulationwhich method comprises administering to a subject an effective amount ofa pharmaceutical composition to bind to the TGF-β, wherein saidpharmaceutical composition comprises a polypeptide comprising theextracellular domain of endoglin and a pharmaceutically acceptablecarrier, thereby treating the pathologic condition caused by cell growthstimulation.
 13. The method of claim 12, wherein TGF-β is TGF-β1. 14.The method of claim 12, wherein TGF-β is TGF-β3.
 15. The method of claim12, wherein the pathologic condition is inflammation, rheumatoidarthritis, inflamed skin lesions, scar tissue formation, lung fibrosis,liver fibrosis, atherosclerosis or glomerulonephritis.
 16. The method ofclaim 12, wherein the subject is a human patient.
 17. A method oftreating a pathologic condition caused by TGF-β regulated inhibition ofcell growth which method comprises administering to a subject aneffective amount of the pharmaceutical composition to bind to the TGF-β,wherein said pharmaceutical composition comprises a polypeptidecomprising the extracellular domain of endoglin and a pharmaceuticallyacceptable carrier, thereby treating the pathologic condition caused byinhibition of cell growth.
 18. The method of claim 17, wherein TGF-β isTGF-β1.
 19. The method of claim 17, wherein TGF-β is TGF-β3.
 20. Themethod of claim 17, wherein the pathologic condition is ulceration orimmune suppression.
 21. The method of claim 17, wherein the subject is ahuman patient.